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Identification of Mutations in 90 of 121 Consecutive Symptomatic French
Patients With a Type I Protein C Deficiency
By S. Gandrille, M. Aiach, and The French INSERM Network on Molecular Abnormalities Responsible
for Protein C and Protein S Deficiencies
By studying the protein C gene of 121 consecutive patients
with symptomatic type I protein C deficiency, we detected
55 different candidatemutations in 90 cases. The mutations,
769’0 of which were missensechanges, were distributed
throughout the gene. More than half the missense mutations involved Cys,Phe,Pro,orGly,
amino acids known
to affect the structure of the polypeptide chain by various
mechanisms. Thus, 409’0 of protein C deficiencies may be
caused by polypeptide chain instability rather than a lack of
expression of the mutated allele; this may also account for
phenotypic heterogeneity. Seventeen
of the 55 different mutations were found in apparently unrelatedfamilies. Halfthe
17 mutations.
French families we studied bore one of these
The wide variety of mutations suggests that both sporadic
casesand a founder effect contribute to the spectrum of
protein C mutations in a given population. The differences
in both unique and recurrent
mutations in French and Dutch
populations-the only large population samples so far studied-support this hypothesis.
0 1995 by The American Society of Hematology.
P
ROTEIN C is a vitamin K-dependent blood coagulation
s~bjects’~.’‘).
Some homozygous (or compound heterozyinhibitor playing an important role in the protein C
gous) subjects with plasma protein C levels below 25% develop purpura fulminans or skin necrosis and intravascular
anticoagulant pathway, as attested to by thrombotic disorders
disseminated coagulation at
while others only dein patients with hereditary deficiencies.“’ Protein C is envelop thrombotic episodes during a d ~ l t h o o d . ~This
* - ~ ~clinicoded by a gene located on chromosome 2, at position 2q13cal heterogeneity could reflect a variety of molecular mechaq21,4-6spanning 11.6 kb’ and comprising 9 exons,’ the first
nisms.
of which is not translated. The intron-exon organization of
When we began this study in 1990, only three mutations
the gene reflects the structural protein domain partition, as
hadbeen associated with protein C d e f i c i e n ~ y . ~Many
~-~~
in the case of other vitamin K-dependent proteins such as
mutations have now been described, and most are associated
factors IX and X and suggests that exons are associated by
with quantitative (type l) deficiencies. Most deficient patients
shuffling.’ Exon I1 encodes the signal peptide, exon 111 the
are heterozygous for a point
About
15 homozypropeptide and Gla domain, exon IV a short helical aromatic
gous22,23,48-53
or compound heterozygous
have
stretch, exons V and VI each encode an epidermal growth
been genotyped.
factor-like domain (EGF l and EGF 2), exon VI1 encodes
With one exception:8 studies to date have involved relathe linker and activation peptides, and exons VI11 and IX
tively small and heterogeneous populations; the representaencode the catalytic site, the substrate hydrophobic pocket,
tive spectrum of mutations is only known in The Netherbinding sites for factor Va,”.” and an exosite.I2The protein
lands.28
is synthesized as a single 462-amino acid chain that underWe studied 121 consecutive symptomatic French patients
goes several posttranslational modifications, comprising
to establish the molecular basis of quantitative protein C
cleavage of the signal peptide and propeptide, y-carboxyldeficiencies and to see whether such information may help
ation of the 9 Glu of the Gla domain, p-hydroxylation of
various phenotypes associated with such abAsp 7 1, and cleavage of the linker peptide ( L y ~ ’ ~ ~ - - A r g ’ ~to~ )understand
,
normalities.
giving rise to a protein comprising one light and one heavy
chain linked by a disulfide bridge.”
MATERIALS AND METHODS
Protein C deficiency is inherited as an autosomal dominant
Patients.
Plasma
and DNA samples were obtained from 70 nortrait and has variable penetrance: heterozygous patients with
mal subjects and 280 subjects belonging to 121 consecutive families
plasma protein C levels between 25% and 70% of normal
with a quantitative (type I) protein C deficiency. The deficient subare either symptomatic (1 of 16,000 in the general populajects were recruited between February 1990 and June 1993 by the
tioni4) or asymptomatic (1 of 200 to 1 of 500 of normal
French Network “Molecular Abnormalities Responsible for Protein
From INSERM U 428, Unite‘de Formation et de Recherche (UFR)
des Sciences Pharmaceutiques et Biologiques. Paris, France.
Submitted November 30, 1994; accepted May 31, 1995.
Supported in part by the Institut National de la Sante‘ et de la
Recherche Mkdicale (INSERM), bygrantsfrom
the Association
Frangaise contre le3 Myopathies (AFM), and Stago Laboratories.
Address reprint requests to S. Gandrille, PhD, INSERM iJ.428,
UFR des Sciences Pharmceutiques et Biologiques, 4, Avenue de
I’Ohservatoire, 75006 Paris, France.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/9.5/8607-0036$3.00/0
2598
C and Protein S Deficiencies” on behalf of INSERM in 21 centers
listed in the Appendix.
In most cases, the propositi and their relatives were referred to
our group after one of these specialized centers had diagnosed a
type I protein C deficiency on the basis of repeated blood sampling.
The general strategy was to screen patients with unexplained thrombosis with a coagulation assay (Staclot Protein C, Diagnostica Stago,
Asnitres, France). In patients with protein C levels 570% (a lower
limit admitted by all the members of the network), an amidolytic
assay (Stachrom Protein C, Diagnostica Stago) was performed in
most centers to confirm the diagnosis. To distinguish between type
I and type I1 deficiencies, protein C levels were measured using an
immunoenzymatic assay (Asserachrom Protein C, Diagnostica
Stago) or a Laurel1 assay. In this assay, the lower limit was also
70%, and a ratio of activity to antigen level 2 0 . 8 was considered
to reflect a type 1 deficiency.
8lood, v01 86, No 7 (October l), 1995: pp 2598-2605
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PROTEIN C GENE MUTATIONS
Whenever possible, family studies were undertaken to check that
the genomic abnormalities cosegregated with protein C deficiency.
A totalof 280 subjects,amongwhom 121 werepropositi with
type I protein C deficiency, were included in the study. The mean
116 propositiwhowerenotunderoral
protein C levelsforthe
anticoagulanttherapywere
49.4% 2 15% in theclottingassay,
51.3% 2 14.5% in the amidolytic assay, and 50.8% ? 14% in the
immunologicassay.Thereproducibilitiesofthethreedifferent
plasma assays comprised between 7% and 10%.
Blood samples. Venousblood was collected in evacuated tubes
containing 0.11 moVL sodium citrate, and plasma was kept frozen
until use.
Blood was collected in ethylene diamine tetracetic acid for DNA
studies and kept at 4°C. Leukocytes were isolated within 48 hours
and stored frozen until DNA extraction as described by Bell et
Computer analysis. We used a Dell 486DX33 computer with a
math coprocessor running the MELT 87 and SQHTX MELTMAP
programss9 generously providedby L. Lerman (Massachusetts Institute of Technology, Cambridge).
Materials and methods. All thematerials and methodsusedto
locate and identify mutations (primer sequences, annealing temperatures, size determination of PCR products, and optimal denaturing
gradientgelelectrophoresis(DGGE)conditionsdeterminedusing
the computer programs indicated above, as well
as sequencing conditions) have been described in a previous study."
RESULTS
All the coding sequences and intron-exon junctions of the
gene were studied. Their cumulated length is 2,414 bp, ie,
all coding base pairs, and 22% of the entire protein C gene
sequence.
We detected 113 sequence variations by screening 191
individuals (121 propositi and 70 controls) by means of
DGGE. Ninety-three of these variations were detected in 90
propositi and were presumably deleterious mutations (Tables
1 and 2); twenty-one were probably silent mutations (Table
3), as they were detected in normal subjects, did not change
the encoded amino acid, were located in intronic regions, or
did not cosegregate with the deficiency.
The 90 propositi with protein C sequence variations bore
55 different mutations. The sequence variations detected in
the coding sequences are listed in Table 1, those affecting
the splice site sequences in Table 2, and rare DNA polymorphisms in Table 3.
Presumed deleterious mutations. Five frameshift mutations were identified in exons VI, VII, and IX in six unrelated
patients. Insertions between nucleotides 336313364 or 33641
3365 (1 bp, C), nucleotides 613916140 (2 bp, TT),and nucleotides 8796 to 8802 (1 bp, G) gave rise to stop codons
at codons 119,156, and 382, respectively. A deletion of
nucleotides 8485-8486 or 8486-8487 (2 bp, AC or CA) was
detected in two apparently independent families, and gave
rise to a stop codon at position 329. A deletion of a single
C among six cytosine nucleotides at position 8950 to 8955
abolished the natural stop codon at codon 420 and created
a new stop codon at position 462, theoretically engendering
a protein with an extra 42 amino acids. However, immunoblotting with an antiprotein C antibodyWfailed to detect
protein C variants with an abnormal length.
One patient and two normal subjects bore a 5-bp deletion
2599
(CTGGA) in intron g. This deletion occurred in a tandem
repeat sequence CTGGA-CTGGA in the gene of the general
population.
Only three substitutions creating a new stop codon were
detected. They affected codons 132, 157, and 306 and were
detected in 2, 4, and 1 families, respectively. All occurred
on CpG dimers.
We identified four splice junction sequence changes in
splice donor sites of introns b, e, f, and h (Table 2). Three
were single base substitutions at position + l , +4, or +5
(nucleotides 7257, 74, or 3222, respectively) of the consensus splice donor site sequence. The fourth was a deletion of
5 bp in intron f, deleting nucleotides +2 to +6 and thus
destroying the consensus splice site sequence. Family studies
showed that the mutation occurred de novo.& In addition,
two patients (No. 080 and No. 113) had a single base substitution in exon V, at position -2 of the splice donor site.
A total of 42 different missense mutations were identified
in 70 patients. Table 1 shows their wide heterogeneity. Surprisingly, we found a -5 Arg to Trp mutation in one patient
with a type I deficiency; indeed, this has been described as
the only nucleotide change in the coding region in a patient
with a type I1 deficiency.m Similarly, the Asp 35 to Gly
mutation was also detected in a patient with a type I1 deficiency (unpublished results).
Whenever possible (51 of 90 cases), cosegregation of the
genomic abnormality with the plasma protein C deficiency
was checked by studying the families (Tables 1 and 2).
Rare DNA polymorphisms. A total of 14 DNA sequence
polymorphisms were identified. Six were located in introns
and were identified in normal subjects (nt -1394, G>A; nt
61 17,C>T; deletion of nt 7086 to 7090), did notcosegregate
with a deficiency (nt -1378, G>C; nt -26, C>A), or were
associated with a detrimental mutation in the coding region
(nt 77, C>G&).
The other polymorphisms were located in coding regions
and did not change the encoded amino acid (nt 3381, G>A;
nt 6217, C>A; nt 8513, C>T; nt 8750, T>C; nt 8759, C>T;
and nt 8801, G>A), with the exception of mutation Met 343
to Ile (nt 8744, G>T), detected in a subject with normal
protein C levels (86%, 78%, 90% of antigen, amidolytic
activity, and anticoagulant activity, respectively), and Gly
370 to Ser (nt 8823, G>A), which did not cosegregate with
the deficiency.
Relationship between genotypes and plasma phenotypes.
The 121 subjects recruited for this study were symptomatic
patients referred to specialized centers for unexplained
thrombosis. The diagnosis of hereditary protein C deficiency
was made after clinical and laboratory investigations, as described in Materials and Methods.
The genomic analysis confirmed the diagnosis in 90 of
121 propositi (74%). Most (82, 68%) had protein C levels
between 14% and 60% (coagulation assay), among whom
75 were heterozygous for a protein C gene mutation, 3 had
a confirmed (No. 045) or probable double mutated allele
(No. 078 and 082, see below), 2 were homozygous (No. 0 4
and 057), and 2 were compound heterozygous (No. 019 and
080). In these latter four patients, protein C levels in the
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2600
GANDRILLE ET AL
Table 1. Mutations in the Coding Regionr of the Protein CGene of D.fi&ent SubjNucleotide’
of
Amino
No.Acid Change
-
-40, Q + P
-34, L
P
-5, R - W §
-3, R + C
15.R-Q
35, D G§
40, F L
47, G C
50, C + R
54, P - S
67, G D
76, F L
107, H P(
116, R - W
124, Y C
132, Q -+ Stop
L#
143, R
157, R Stop
168, P L
169, R W
178, R - W
178. R + Q
197, G E
209. A V
220, L P
223, L F
230, R -+ C
232, E K
258, I R55
267, A -,T
270, S P
279, P L
286, R C
292. G + S
293, Q H
297. V M
298, T K
298, T -+ M
306, R Stop
314, R + H
318. L F
323, I F
327, P L
335, M
T
361, G R
363, P Ann
389, N K
403, I L
403, I M
414. Q -+ R##
8.A-C
26.T-C
1375, C T
1381, C T
1433. G A
1493, A G
2971, C - A
3082. G T
3091, T C
3103, C T
G 3143.
A
3169, T -+ C
3363/4, Ins C
3390, C T
3415, A G
3438, C T
6139, Ins TT
6182, C T
6216, C T
6218, C T
6245, C T
A
G 6246,
7176, G + A
7212, C T
7245, T C
7253. C T
8403. C T
8409, G A
Del CA or AC 8485/6 or 8486i7
8514, G - A
8523, T C
8551, C T
8571. C T
8589, G A
8594, G C
8604. G A
8608,C A
8608, C T
8631, C T
8656, G -+ A
8667, C T
8682, A T
8695, C T
8719, T C
8796, G C
Ins G 8796/7/8/9 or 8000/1
8882, C A
8922. A C
8924, C G
Del C at 8950/1/2/3/4/5
------
-----
Families
1
1
-+
-----------+
+
----
+
--
1
109
1
1
1
1
+
3
---
------
-+
5
1
-+
1
1
1
4
1
1
1
2
1
1
2
2
1
2
1
056, 2
1
4
5
1
1
8
4
1
1
4
2
1
2
1
1
1
3
3
1(31
3
1
1
1
1
1
1
l
1
1
(2)
(4)
2 (2)
8 (28)
1 (1)
1 (1)
3 (4)
6 (6)
(31
l
1
1
1
1
1
Identity No.*
cXJy,t
(1)
(2)
1 (1)
4 (4)
2 (2)
2 (2)
9 (10)
l(1)
3
(1)
7 (9)
5 (5)
1Il)
(1)
18 (29)
6 (8)
(1)
l (1)
14 (26)
10 (20)
l(1)
4 (7)
13)
1 (5)
2 (2)
(3)
7 (7)
3
(9)
2 (2)
l(1)
(1)
1 (1)
4 (5)
2 (2)
2 (3)
4 (71
l(1)
1 12)
(1)
2 (2)
6 (12)
1
026
025
082
022,033,043, 076
IO031
078
046
097,098
103
064
048, 110
104, 08011
107
111,118
108
088
10061.10071,
10051,
085
008.024,039,044**, 074
052
02 1
10101,
10091.
037,045tt.
062,073,
061,053,
038,040.041,092
045tt
091
027,019*$, 035,036
060, 117
023
016,055
094
04911l1
087
032, 050,075
057’”. 058,086
030
014, 031,051
042
018
015
08011
063
059
017
020
089
013
093
052
019**
090
The one-letter code used for the emino acids is as follows: A, Alanine; C, Cysteine; D, Aspartic acid; E, Glutamic acid; F, Phenylalanine; G, Glycine; H, Histidine; l,
Isoleucine; K, Lysine; L, Leucine; M, Methionine; N. Asparagine; P, Proline; Q, Glutamine; R, Arginine; S,Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.
Nucleotide numbering according to Foster et al.’
t xly): x = number of heterozygoushomozygous subjects and y = total number of propositus’ family members tested.
Patients are identified according to the database numbering,’’ ie. PC-33-X. Patients already listed in the database are indicated between brackets.
5 The -5 R -,W mutation has already been identified i n a patient with a type II deficiancy.m We have also identified a 35 D G mutation in a patient with a type 1
I
deficiency (unpublished results).
I due to a 9 R C mutation).
I( Double mutant IF76 U314 R H) i n a compound heterozygous subject with type I and type II deficiency (type 1
Frameshift at position 107 and stop codon at position 119.
#Frameshift at position 143 and stop codon at position 156.
** Homozygous subject.
tt Double mutant I209 A V1178 R 0)in a patient heterozygous for a type I deficiency.
*$ Compound heterozygote subject for type I deficiency (L223 F and 403 I MI.”
55 Frameshift at position 258 and stop codon at position 329.
I( I(De novo mutation.“
T
!IFrameshift at position 363 and stop codon at position 382.
##Frameshift at position 414, which suppresses the natural stop codon at position 420. New stop codon at position 461.
*
-
-+
-+
+
n
+
-+
-
-+
antigen, amidolytic, and coagulant assays were respectively
nd/15%/ndinpatient 044, 22%/16%/16%inpatient 057,
16%/15%/26%in patient 019, and 47%/56%/14%in patient
080 (compound heterozygote for typeI and type I1 deficiencies) .
Among the 31 propositi with no identified abnormality in
the protein C gene coding sequence, 15 had protein C levels
between 60% and 70% (coagulationassay). In this latter
group, familial studies was either impossible (10 propositi)
or did not show a protein C deficiency in the relative ex-
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2601
PROTEIN C GENE MUTATIONS
Table 2. Mutations Affecting Splking of the Rotein c Gene
of Deficient Subjects
Families
Nucleotide
IntronNumbering'
74, A "* G
3216, C + T
3222, G + A
Del 3455-3459
7257, G + T
~
No. of
b, splice donor
exon 5,91 R -t R §
e, splice donor
f, splice donor
h, splice donor
1
2
1
1
1
2
3
1
x(y)t
Identity
No.*
6 (6)
(2)
(4)
(5)
l(1)
054
080, 113
047
10511
034
~~
Nucleotide numbering according to Foster et al.'
t x(y): x = number of heterozygous/homozygous subjects and y =
total number ofpropositus' family members tested.
Patients are identified according to the database numbering?' ie,
PC-33-x.
5 Mutation also affecting the splice donor site of intron e.
1) De novo mutation.
*
plored (five propositi). It is thus possible that some of the
31 patients have low protein C levels due to causes other
than a genetic defect.
DISCUSSION
Genomic analysis of 121 consecutive French patients with
a type I protein C deficiency allowed us to detect 55 different
mutations in 90 patients (74%).Large deletions or insertions,
which cannot be detected by our approach, remain to be
sought in the 31 patients in whom we did not detect point
mutations.
These mutations cosegregated withtheprotein C deficiency when family studies were possible (51 families) and
none were found in the protein C gene of 70 controls with
normal protein C levels. Thus, the 55 mutations identified
in this series probably have a detrimental effect on the expression of the corresponding allele.
Eight changes were nonsense mutations or frameshifts,
which generate a truncated polypeptide chain or alter the
reading frame, generating stop codons or aborted proteins,
respectively. Frameshifts frequently introduce premature
stop codons, but one frameshift in this series shifted the stop
codon from codon 420 to codon 462, theoretically giving
rise to an elongated protein. Immunoblotting failed to show
an abnormal protein in the patient's plasma, suggesting that
the polypeptide, if synthesized, was not secreted. It is noteworthy thatall the patients, but one (No. 016), with nonsense
or frameshift mutations had protein C levels below 60%.
Splice site mutations (five in our series) are deleterious,
as theydestroythe conserved positions of the consensus
splice site sequences and impair mRNA splicing, a mechanism known to impair translation. Two patients (pedigrees
No. 080 and 113) were heterozygous for a C and T (nucleotide 3216) at position -2 of the donor splice site of exon
V. Even if this apparently silent mutation did not change the
amino acid (Arg), a detrimental effect due to abnormal
mRNA stability or to exon skipping cannot be ruled out.
The latter was recently described in the case of mutations
occurring at position - 1 of the donor splice site of exon VI1
of the protein C gene36 and at position-3 of those of exon
28 of the GP IIb-IIIa gene.61Studies of mRNA structure and
expression will help clarify this issue. The detrimental effect
of the 42 missense mutations is more difficult to ascertain.
Some of the mutations involvedaminoacidswithkey
positions; for example, Gln -40 and Leu -34 form part of
the signal sequence of protein C, which is necessary for
translocation of the neosynthesized chain into the endoplasmic reticulum"~63; Gln -40 belongs to the n-region, and its
Table 3. List of Rare DNA Polymorphisms Detected in Patientsor Normal Subjects
No. of Subjects Bearing
the Polymorphism
Normal
Deficient
Subjects
Subjects
AcidAmino
Nucleotide
-1394, G + A
-1378, G "*C
-26, C + A
77, C -t G
Comments
None
None
2
None
None
3
1
3381, G "*A
6117,C+T
C 6217, + A
Del 7086 to 7090
8513, C "* T
112, E E
None
168,P"*P
1None
266, P P
8744, G -, T
8750, T -t C
8759,
C
T
8801, G -,A
8823, G -, A
343, M + I
345, c c
348, I I
362, G -, G
370, G -, S
-+
1
1
+
1
1
2
l
-+
-+
1
intron
1
1 1
1
+
intron
In
a
Inintron a. In a patient with a type II deficiency. No cosegregation withthe
deficiency
intron
In
a. No cosegregation
the
with
deficiency
In intron b. Mutation detected in patient(327,
017
P 4 L, this study) and
cosegregating with the deficiency
In patient(91,
113
R 4 R, this study)
In
f
In intron g
Homozygous polymorphism in a patient with a type II heterozygous plasma
deficiency phenotype
In patient 034 (nt 7257, G -t T)
Polymorphism detected in a patientwith a type II deficiency phenotype
1
1
Mutation detected in a type I deficient family, butno cosegregation with
the deficiency
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2602
"
-
GANDRILLE ET AL
E6F2
V
VI
VI1
wdrophobk
A
c
h
tn
*
a
Adhrdkn
Eff1
a
pocket
1 1
a
I
IX
VI11
I
GLA-domaln
[:l,: i.
Y
mm
-42
+G
- l
mm...@.+
+
mm+
m
A
,vmeu
l00
m.
...m
.@m.
Nonsense
*IYlrsenre
VAFramoahift
+Splice
t
m. . m
m.
m m e.w*e$e-200
Amlnoacklnumbor
substitution by a hydrophobic and helix-breaking Pro may
diminish the stability of the domain; Leu -34 is part of the
hydrophobic core (h-region) and belongs to the polyleucine
sequence. Hydrophobicity does not change when Leu is replaced by Pro, but the helix-breaking effect may again impair
the signal peptide conformation and, thus, translocation of
the polypeptide chain.
Other mutations affected amino acids highly conserved in
protein C of mammalian species, such as Pro 54, 168, 279,
and 327, or in protein C of mammals and other vitamin Kdependent proteins (factors 11, VII, IX, and X)"69 such as
substitutions affecting Gly 67, 197, and 361, attesting to the
structural importance of these amino acids.
It is noteworthy that 62% (26 of 42) of the missense
mutations in this series involved only 5 of the 20 existing
amino acids (Cys, Phe, Trp, Gly, and Pro). These 5 amino
acids are known to affect the structure of the polypeptide
chain by various mechanisms. Mutations affecting Cys or
introducing new Cys residues in the sequence may disrupt
the secondary structure or impair the folding or intracellular
trafficking by homodimerization or heterodimerization. Mutations affecting Phe, Trp, and Gly or introducing these residues disturb the structure of the polypeptide chain by a difference in bulk between the normal and the mutated amino
acid. Finally, mutations involving Pro may disrupt the structure of the polypeptide chain by modifying the rigidity of
the chain through a helix- or &sheet breaking effect.
On the basis of a three-dimensional model, Greengard et
a17' recently proposed explanations for the detrimental effect
of six mutations detected in this series (Arg 169 to Trp, Arg
178 to Trp or Gln, Ala 267 to Thr, Thr 298 to Met, and Ile
403 to Met).
We have only indirect arguments for a detrimental effect
of mutations Ala 209 to Val, Gln 293 to His, Val 297 to
Met, Thr 298 to Lys, Arg 314 to His, Met 335 to Thr, Asn
389 to Lys, and Ile 403 to Leu. All these amino acids are
conserved among nine mammalian protein C species and are
thus structurally important. None were detected in the panel
of 70 normal subjects. Site-directed mutagenesis and expression studies could help to establish whether or not these
mutations are detrimental.
The location of the mutations is shown in Fig 1. They
a.
a m
I
N
,:A ,
3 0 0structural or functional
4 0 0 and
Fig 1. Location of the mutations identifiedin the 90 proporiti.The exonintron partition
and
the conetapondance betweenexonr
domains of tho protein are indicated on the upper barrel.
occurred throughout the sequence, although two domains,
encoded by exon VI1 and the 5' half of exon IX, appear to
be preferential sites for mutations. Such a large spectrum of
mutations in patients living within the same geographic area
suggests a high rate of de novo mutations. The finding of
two sporadic cases in a series of 40 families studied4' supports this hypothesis. Subjects heterozygous for a protein
C gene mutation have late clinical expression38or remain
asympt~matic,'~,'~
which allows their genomic abnormality
to be transmitted to their descendents. This probably explains
why17 mutations are apparently recurrent and account for
type I protein C deficiencies in 54 (60%) of the 90 propositi
bearing a point mutation of the protein C gene coding sequence.
The spectrum of recurrent mutations in the French population is completely different from that found in The Netherlands, where the most frequent mutations affect Gln 132 and
Arg 230, rather than Arg178 and Pro 168 in the French
population. Interestingly, the Leu 223 to Phe mutation, preferentially found in the north of France,56 has also been described in Dutch patients." A founder effect probably explains the differences between these two populations.
Among the French patients with point mutations, 76%
bore a missense mutation, the remainder being accounted
for by frameshifts and nonsense or splice site mutations.
These results are consistent with those reported in the
literature, particularly those in the database?' The proportion
of missense mutations is very high and the spectrum very
wide, making the molecular basis for protein C deficiencies
very similar to that of factor IX deficiencies causing hemophilia B." However, the mode of inheritance of protein C
deficiency is much more complex than that of hemophilia
B.
Although the probability of compound heterozygosity and
homozygosity is low, we identified complex genotypes in
four subjects (4.4%).Two were compound heterozygotes for
type I deficiencys6or for a combined type I and I1 deficiency.
In the latter case, the patient had an allele bearing an Arg 9
to Cys mutation (responsible for type I1 deficiency), whereas
the other allele was doubly mutated with Phe 76 to Leu and
Arg 314 to His substitutions, both of which are known to
be associated with type I deficiency in heterozygous sub-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2603
PROTEIN C GENE MUTATIONS
j e ~ t s . ~ We
~ . ” identified Pro 168 to Leu and Gly 292 to Ser
mutations in both homozygous (No. 044 and 057) and symptomatic heterozygous patients. Mutations Ala267 to Thr
and Arg 286 to Cys, observed in symptomatic heterozygous
propositi, had already been described in homozygous patient~.~’”~
Thus, parents of homozygous subjects bear mutations that can lead to thrombotic complications, even in heterozygous subjects.
We also observed a double mutation in patient No. 045,
who had an alieie bearing Ala 209 to Val and Arg 178 to
Gln substitutions. This doublymutant genotype probably
explains the phenotype of two subjects with Arg -5 to Trp
or Asp 35 to Gly mutations, which are associated with type
I1 deficiencies in other patients@(unpublished results). The
allele bearing these mutations might also bear a large deietion or insertion, precluding the expression of the allele and
giving rise to a type I deficiency.
The wide variety of missense mutations may explain the
heterogeneity of the plasma phenotype and clinical expression of protein C deficiency. Recent reports of mutations in
homozygous s u b j e ~ t s ~ and
~ - ‘ ~mutation expression experim e n t ~ ~ ’show
~ ’ ~ that some missense mutations are associated
with a total absence of gene expression, while others permit
partial gene expression and secretion of a small quantity of
protein. T h i s is reminiscent of n,-antitrypsin deficiencies, in
which some mutations cause a null phenotype and others a
plus phenotype.
The absence of mutations in 3 1 (24%)patients underlines
the difficulty of diagnosing hereditary protein C deficiency.
Large gene rearrangements have never beenwell documented and, if they exist, could not explain the low protein
C levels observed in all 31 propositi. Our strategy to detect
point mutations is basedon DGGE, the most sensitive
screening method for point mutation^.'^ It was carefully set
up and tested with known mutations,* making it unlikely
that nucleotide substitutions were missed in as many as 24%
of thepatients. Half the propositi with no mutations had
protein C levels between 60% and 70% and thus might be
normal. The overlapping values observed in normal and heterozygous subjects:’ as well as intraindividual fluctuations
in protein C concentrations,16suggest that a sizeable proportion of patients in our series were misclassified. Genomic
analysis is thus a valuable diagnostic tool in some cases of
ambiguous plasma phenotypes.
ACKNOWLEDGMENT
We thank Drs L.S. terman and K. Silverstein, who lundly provided us with the computer programs MELT 87 and SQHTX.
APPENDIX
List of participating laboratories: Dr J.F. Abgrdl, HBpitaal Augustin Morvan, Brest, France; Dr M. Alhenc-Gelas and Dr J. Emmerich, H6pital Broussais, Paris, France; Dr M.F. Aillaud and Pr I.
Juhan-Vague, CHU La Tirnone, Marseille, France; Dr C. Boinot,
CHU La Milehie, Poitiers, France; Dr M.H. Denninger, HBpital
Beaujon, Clichy, France; Dr M. Dreyfus, Hapital du Kremlin-BicCwe, Kremlin BicCtre,France; Dr E. Dupuy and Dr P. Molho-Sabatier,
HBpital LariboisiBre, Paris, France: Dr A.M. Fischer, H6pital Laen-
nec, Paris, France; Dr F, Forestier, Institut de Putriculture, Paris,
France; Dr M. Gouault-Heilmann, Hapita1 H. Mondor, Ckteil,
France; Dr L. Houbouyan, HBpitaI Ambroise Park, Boulogne,
Fmnce; Dr P. HourdillB, HBpital du Haut Leveque. Pessac, France;
Dr B. Jude, Hbpital Cardiologique, Lille, France; Dr A. MichaudMallet, CRTS, St Laurent du Var. France; Dr P. de MoerIoose and
Dr G. Reber, HBpital Cantonal Universitaire, Genkve, Suisse; Dr M.
Pommereuil, CHU Pontchaillou, Rennes, France; Dr J. Reynaud,
H6pital Nord, St Etienne, France; Dr 3. Roussi, HBpital Raymond
Poincarrk, Garches, France; Dr J. Sampol, HBpital La Conception,
Marseille, France; Dr F. Sit, CRTS-HBpitai Purpart, Toulouse,
France; and Dr P. Toulon, HBpital Cochin, Paris, France.
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1995 86: 2598-2605
Identification of mutations in 90 of 121 consecutive symptomatic
French patients with a type I protein C deficiency. The French
INSERM Network on Molecular Abnormalities Responsible for Protein
C and Protein S deficiencies
S Gandrille and M Aiach
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